Fig. 2

TNFα mediates loss of CD31⁺Sca-1^high vessels in the BM niche of FLT3-ITD+ AML. a Levels of TNFα (pg/ml, left) in the BM and blood analyzed by Luminex assay (n = 10 mice per group) and levels of TNFα mRNA (right) in the BM CD45⁺ cell subpopulations analyzed by Q-RT-PCR (n = 4–7 mice for each population) from wt and Mll^PTD/WT/Flt3^ITD/ITD AML mice. b and c Number of ECs from normal wt mice after in vitro exposure to murine recombinant (mr) TNFα (1 ng/ml) or vehicle for 96 h (b) and frequency of Sca-1^high EC subfraction after in vitro exposure to mrTNFα (0, 0.2, 1 and 10 ng/ml) for 96 h (c), analyzed by flow cytometry. One of the three independent experiments with similar results is shown. d–f Normal wt mice treated with vehicle or mrTNFα (1 µg/day, ip, 3 weeks) were evaluated by flow cytometry (n = 4 mice per group) for frequencies of BM ECs (d) and Sca-1^high and Sca-1^low EC subfractions (e, left, representative plots; right, aggregate results) and by tibial CD31 (FITC) and Sca-1 (PE) IF staining (f, left) and quantification (f, right; n = 3 mice per group) of CD31⁺Sca1^high EC-lined vessels. For f: yellow arrows indicate CD31⁺Sca-1^high EC-lined vessels; white arrows indicate CD31⁺Sca-1^low EC-lined vessels. Scale bars represent a size of 100 µm. g and h Diseased Mll^PTD/WT/Flt3^ITD/ITD AML mice treated with IgG or anti-TNFα Abs (1 mg/day, ip, 4 times/week, 3 weeks; n = 4 mice per group) were evaluated by flow cytometry for frequencies of BM Sca-1^high and Sca-1^low EC subfractions (g, left, representative plots; right, aggregate results) and by tibial CD31 (FITC) and Sca-1 (PE) IF staining (h left) and quantification (h right) of CD31⁺Sca1^high EC-lined vessels. For h: yellow arrows indicate CD31⁺Sca-1^high EC-lined vessels; white arrows indicate CD31⁺Sca-1^low EC-lined vessels. Scale bars represent a size of 100 µm. Results represent mean ± SEM. Significance values: *p < 0.05; **p < 0.01; ***p < 0.001; ns not significant